Tuberculosis is now second behind AIDS, as the World's most deadly microbial infection. However, a major fraction of AIDS patients die of mycobacterial infections, including TB. The TB problem is aggravated by the growing prevalence of drug-resistant TB, and especially multi-drug resistant (MDR) TB which cannot be treated with the front-line antibiotics for Mycobacterium tuberculosis. It is therefore important that targets be identified for development of new drugs for treatment of MDR TB. Suitable target enzymes should have biochemical functions essential for mycobacteria but with no similar function in mammals making it likely that drugs can be developed that will not lead to adverse reactions in humans. They should have well-defined assays suitable for screening of potential drugs. The proposed research elucidates the biochemistry associated with the production and utilization of the antioxidant thiol known as mycothiol. Mycothiol is produced only by mycobacteria, and other actinomycetes, and is not found in animals. The key genes for mycothiol biosynthesis have recently been identified and provide important potential new drug targets. Studies of MSH-deficient mutants indicate that mycothiol metabolism is involved in protecting against oxidative damage and in the detoxification of antibiotics, including one first-line TB drug. Although not essential for the laboratory culture of the model organism Mycobacterium smegmatis, current evidence suggests that mycothiol may be required for survival of M. tuberculosis in an oxygen rich environment. The present studies will determine the extent to which mycothiol is essential for survival of M. tuberculosis, will define the biochemistry involved in the first key step of mycothiol biosynthesis, and will determine how the biosynthesis of mycothiol is regulated. Methods used include new analytical and enzyme assays developed in these laboratories as well as established protocols in biochemistry and molecular biology. The results obtained will provide a key test of the suitability of mycothiol biosynthesis as a target for new TB drugs and will elaborate the biochemistry of a novel class of thiol important to a broad class of soil microorganisms, including most antibiotic producing bacteria.